The goal of the proposed research is to understand molecular mechanisms by which select proteins are targeted to and translocated across specific membranes within the eukaryotic cell. In particular, we propose to employ genetic and biochemical methods to study the localization of the nuclear protein Alpha2 in the yeast Saccharomyces cerevisiae. The molecular basis by which specific proteins accumulate post-translationally in the nucleus is poorly understood. We have previously shown that the Alpha2 protein is indeed a nuclear protein and that the thirteen amino terminal residues of Alpha2 are capable of targeting E. coli Beta-galactosidase (as a Alpha2-Beta-gal hybrid protein) to the nucleus. The following questions remain to be answered. a) What within the amino-terminal thirteen residues of Alpha2 is necessary and sufficient as a localization determinant? b) Is there additional information within Alpha2 (other than the amino-terminal thirteen amino acids) required for or capable of mediating entry into the nucleus? c) What is the machinery in the nuclear envelope that allows passage not only of endogenous proteins but also a large Alpha2-Beta-gal hybrid protein? d) Do known components of the nuclear envelope, e.g. lamin and pore-complex, play a role in nuclear localization? e) Is the translocation of proteins across the nuclear envelope an energy-dependent process -- if so, what is the source of this energy?